Generation of transgenic Nicotiana benthamiana plants conferring resistance against Cucurbit chlorotic yellows virus

[[abstract]]Cucurbit chlorotic yellows virus (CCYV), belonging the genus Crinivirus of the family Closteroviridae, possesses a positive-sense single-stranded bipartite RNA genome, denoted RNA1 and RNA2, encapsidated in separated particles. CCYV infects economic cucurbit crops through vectoring of Bemisia tabaci biotypes B and Q and causes severe yield losses in Asia, Africa and Europe, including Taiwan. Although resistance breeding is a common approach to control plant virus, efficient control measures are still difficult by traditional breeding due to the high divergence of viruses and scarcity of natural resistant resources. Alternatively, the transgenic approach mediated by RNA silencing is the most effective strategy for control of plant viruses. In this study, an untranslatable transgene construct consisting of the conserved regions of RNA-dependent RNA polymerase (RdRp) and heat shock protein 70 homologue (Hsp70h) genes linked with the P22 open reading frame, the suppressor of plant RNA silencing, of CCYV was used to generate transgenic Nicotiana benthamiana plants. Fourteen transgenic lines were challenged with CCYV by transmission of silverleaf whitefly (B. tabaci biotype B) for evaluation of resistance by observation of symptom development for 7 weeks post-inoculation (wpi). Ten lines with different degrees of resistance were obtained and five of them without symptoms were considered highly resistant (HR). CCYV infection could be detected in all challenged resistant plants but the accumulation of viral RdRp transcripts was significantly reduced in the HR lines. Low levels of transgene transcripts coupled with high amounts of small RNAs were detected, demonstrating the resistance is mediated by RNA silencing. Thus, we establish a potential approach that can be applied in cucurbit crops, such as melon and watermelon, for control of CCYV disease